Christiane Emmerich’s research while affiliated with Max Planck Institute for Developmental Biology and other places

One of the most extensive forms of natural genome editing occurs in ciliates, a group of microbial eukaryotes. Ciliate germline and somatic genomes are contained in distinct nuclei within the same cell. During the massive reorganization process of somatic genome development, ciliates eliminate tens of thousands of DNA sequences from a germline genome copy. Recently, we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia . Here, we describe two high similarity paralogous proteins, ICOPa and ICOPb, essential for their genome editing. ICOPa and ICOPb are highly divergent from known proteins; the only domain detected showed distant homology to the WSD (WHIM2+WHIM3) motif. We show that both ICOPa and ICOPb interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative DNA excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOPa and ICOPb are needed for Paramecium's precise genome editing.

Most eukaryotes have one nucleus and nuclear genome per cell. Ciliates have instead evolved distinct nuclei that coexist in each cell: a silent germline vs. transcriptionally active somatic nuclei. In the best-studied model species, both nuclei can divide asexually, but only germline nuclei undergo meiosis and karyogamy during sex. Thereafter, thousands of DNA segments, called internally eliminated sequences (IESs), are excised from copies of the germline genomes to produce the streamlined somatic genome. In Loxodes , however, somatic nuclei cannot divide but instead develop from germline copies even during asexual cell division, which would incur a huge overhead cost if genome editing was required. Here, we purified and sequenced both genomes in Loxodes magnus to see whether their nondividing somatic nuclei are associated with differences in genome architecture. Unlike in other ciliates studied to date, we did not find canonical germline-limited IESs, implying Loxodes does not extensively edit its genomes. Instead, both genomes appear large and equivalent, replete with retrotransposons and repetitive sequences, unlike the compact, gene-rich somatic genomes of other ciliates. Two other hallmarks of nuclear development in ciliates—domesticated DDE-family transposases and editing-associated small RNAs—were also not found. Thus, among the ciliates, Loxodes genomes most resemble those of conventional eukaryotes. Nonetheless, base modifications, histone marks, and nucleosome positioning of vegetative Loxodes nuclei are consistent with functional differentiation between actively transcribed somatic vs. inactive germline nuclei. Given their phylogenetic position, it is likely that editing was present in the ancestral ciliate but secondarily lost in the Loxodes lineage.

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition 1 . All non-standard ciliate genetic codes involve stop codon reassignments 1,2,3 . Two of these codes are ambiguous 1–3 , with “stop” codons either translated or terminating translation depending on their context 2,3 . Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia 4 and an alveolate species from the genus Amoebophrya 5 . Two ambiguous genetic codes in ciliates share translation of UGA “stop” codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons 1,2 . Furthermore, though a UGA-cognate tRNA Cys UCA was reported in Euplotes 6 , a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNA Trp UCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNA Trp CCA ’s, rather than the typical 5 base pair stem tRNAs 7 . New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination 7 . Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing “stop” codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition.

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition 1 . All non-standard ciliate genetic codes involve stop codon reassignments 1,2,3 . Two of these codes are ambiguous 1–3 , with “stop” codons either translated or terminating translation depending on their context 2,3 . Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia 4 and an alveolate species from the genus Amoebophrya 5 . Two ambiguous genetic codes in ciliates share translation of UGA “stop” codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons 1,2 . Furthermore, though a UGA-cognate tRNA Cys UCA was reported in Euplotes 6 , a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNA Trp UCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNA Trp CCA ’s, rather than the typical 5 base pair stem tRNAs 7 . New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination 7 . Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing “stop” codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition.

Ciliates are unicellular eukaryotes with two distinct kinds of nuclei in each cell: transcriptionally active somatic macronuclei (MAC) and silent germline micronuclei (MIC). In the best-studied model species, both nuclei can divide asexually, but only germline MICs participate in meiosis, karyogamy, and development into new MACs. During MIC-to-MAC development, thousands of mobile element relics in the germline, called internally eliminated sequences (IESs), are excised. This genome editing enables IESs to persist by shielding them from somatic natural selection. Editing itself is a costly, time-consuming process, hypothetically maintained by evolutionary addiction. Loxodes magnus and its relatives (class Karyorelictea) are cytologically unusual because their MACs do not divide asexually, but must develop anew from mitotically generated MIC copies every cell division. Here, we report that Loxodes genome development is also unconventional. We found no canonical germline-limited IESs in Loxodes despite careful purification and long-read sequencing of MICs and MACs. The k-mer content of these nuclei overlapped, and indels found by read mapping were consistent with allele variants rather than IESs. Two other hallmarks of genome editing--domesticated DDE-family transposases and editing-associated small RNAs--were also absent. Nonetheless, histone marks, nucleosome and DNA N6-methyladenosine distributions suggest that MACs are actively transcribed and MICs inactive in vegetative Loxodes cells, like other ciliates. Both genomes, not only the MIC, were large and replete with retrotransposon sequences. Given the costs associated with genome editing, we hypothesize that karyorelicteans like Loxodes have lost or streamlined editing during MIC-to-MAC development, and have found a way out of the addictive cycle.

Ciliates are a widespread clade of microbial eukaryotes with the greatest diversity of nuclear genetic codes (at least eight) following a recent addition ¹ . All non-standard ciliate genetic codes involve stop codon reassignments 1,2,3 . Two of these codes are ambiguous ¹⁻³ , with "stop" codons either translated or terminating translation depending on their context 2,3 . Ambiguous genetic codes have arisen not only in ciliates, but also independently in trypanosomatids from the genus Blastocrithidia ⁴ and an alveolate species from the genus Amoebophrya ⁵ . Two ambiguous genetic codes in ciliates share translation of UGA "stop" codons as tryptophan with Blastocrithidia and the Amoebophrya species. tRNA genes with complementary anticodons to reassigned UAA and UAG stop codons have invariably been found in ciliate species that translate these codons 1,2 . Furthermore, though a UGA-cognate tRNA Cys UCA was reported in Euplotes ⁶ , a ciliate genus that translates UGA as cysteine, vexingly, no nuclear genome-encoded tRNA Trp UCA has been found in ciliate species with UGA tryptophan codons. Recently, Kachale et al. provided evidence for UGA translation as tryptophan in Blastocrithidia nonstop and the ciliate Condylostoma magnum using 4 base pair anticodon stem (AS) near-cognate tryptophan tRNA Trp CCA 's, rather than the typical 5 base pair stem tRNAs ⁷ . New tRNA data we report from additional ciliates bolsters this hypothesis. Kachale et al. also hypothesised that a particular amino acid substitution in the key stop codon recognition protein, eRF1 (eukaryotic Release Factor 1), favours translation of UGA as tryptophan instead of termination ⁷ . Contrary to Kachale et al, we propose such substitutions favouring reduced eRF1 competition enhancing 'stop' codon translation do not need to occur concomitantly with tRNA alterations or acquisitions to evolve new genetic codes via stop codon reassignment. We report multiple instances of the substitution investigated in Kachale et al. 2023 that have not led to UGA translation, and multiple ciliate species with UGA tryptophan translation but without the substitution, indicating it is not necessary. Consistent with the ambiguous intermediate hypothesis for genetic code evolution, experimental evidence and our observations suggest continued potential ciliate eRF1-tRNA competition.

Chromatin remodeling is required for essential cellular processes, including DNA replication, DNA repair, and transcription regulation. The ciliate germline and soma are partitioned into two distinct nuclei within the same cell. During a massive editing process that forms a somatic genome, ciliates eliminate thousands of DNA sequences from a germline genome copy in the form of internal eliminated sequences (IESs). Recently we showed that the chromatin remodeler ISWI1 is required for somatic genome development in the ciliate Paramecium tetraurelia. Here we describe two paralogous proteins, ICOP1 and ICOP2, essential for DNA elimination. ICOP1 and ICOP2 are highly divergent from known proteins; the only domain detected showed distant homology to the WSD motif. We show that both ICOP1 and ICOP2 interact with the chromatin remodeler ISWI1. Upon ICOP knockdown, changes in alternative IES excision boundaries and nucleosome densities are similar to those observed for ISWI1 knockdown. We thus propose that a complex comprising ISWI1 and either or both ICOP1 and ICOP2 are needed for chromatin remodeling and accurate DNA elimination in Paramecium.

Massive DNA excision occurs regularly in ciliates, ubiquitous microbial eukaryotes with somatic and germline nuclei in the same cell. Tens of thousands of internally eliminated sequences (IESs) scattered throughout the ciliate germline genome are deleted during the development of the streamlined somatic genome. The genus Blepharisma represents one of the two high-level ciliate clades (subphylum Postciliodesmatophora) and, unusually, has dual pathways of somatic nuclear and genome development. This makes it ideal for investigating the functioning and evolution of these processes. Here we report the somatic genome assembly of Blepharisma stoltei strain ATCC 30299 (41 Mbp), arranged as numerous telomere-capped minichromosomal isoforms. This genome encodes eight PiggyBac transposase homologs no longer harbored by transposons . All appear subject to purifying selection, but just one, the putative IES excisase, has a complete catalytic triad. We hypothesize that PiggyBac homologs were ancestral excisases that enabled the evolution of extensive natural genome editing.

During their development following sexual conjugation, ciliates excise numerous internal eliminated sequences (IESs) from a copy of the germline genome to produce the functional somatic genome. Most IESs are thought to have originated from transposons, but the presumed homology is often obscured by sequence decay. To obtain more representative perspectives on the nature of IESs and ciliate genome editing, we assembled 40,000 IESs of Blepharisma stoltei , a species belonging to a lineage (Heterotrichea) that diverged early from those of the intensively studied model ciliate species. About a quarter of IESs were short (<115 bp), largely nonrepetitive, and with a pronounced ~10 bp periodicity in length; the remainder were longer (up to 7 kbp) and nonperiodic and contained abundant interspersed repeats. Contrary to the expectation from current models, the assembled Blepharisma germline genome encodes few transposases. Instead, its most abundant repeat (8,000 copies) is a Miniature Inverted-repeat Transposable Element (MITE), apparently a deletion derivative of a germline-limited Pogo-family transposon. We hypothesize that MITEs are an important source of IESs whose proliferation is eventually self-limiting and that rather than defending the germline genomes against mobile elements, transposase domestication actually facilitates the accumulation of junk DNA.

Loxodes is one of the best ecologically characterized ciliate genera with numerous intriguing physiological abilities, including gravity-sensing organelles and nitrate respiration. However, these cells have been considered challenging to cultivate in bulk, and are poorly preserved by conventional fixatives used for fluorescence microscopy. Here we describe methods to grow and harvest Loxodes cells in bulk with liquid soil extract medium, as well as a new fixative called ZFAE (zinc sulfate, formaldehyde, acetic acid, ethanol) that can fix Loxodes cells more effectively than buffered formaldehyde or methanol. We show that ZFAE is compatible with immunofluorescence and the nuclear stain DAPI. Loxodes is thus now amenable to long-term maintenance, large-scale growth, and modern cell biology investigations of monoclonal strains in laboratory conditions.